Psychromonas boydii sp. nov., a gas-vacuolate, psychrophilic bacterium isolated from an Arctic sea-ice core.

A gas-vacuolate bacterium, strain 174(T), was isolated from a sea-ice core collected from Point Barrow, Alaska, USA. Comparative analysis of 16S rRNA gene sequences showed that this bacterium was most closely related to Psychromonas ingrahamii 37(T), with a similarity of >99 %. However, strain 174(T) could be clearly distinguished from closely related species by DNA-DNA hybridization; relatedness values determined by two different methods between strain 174(T) and P. ingrahamii 37(T) were 58.4 and 55.7 % and those between strain 174(T) and Psychromonas antarctica DSM 10704(T) were 46.1 and 33.1 %, which are well below the 70 % level used to define a distinct species. Phenotypic analysis, including cell size (strain 174(T) is the largest member of the genus Psychromonas, with rod-shaped cells, 8-18 microm long), further differentiated strain 174(T) from other members of the genus Psychromonas. Strain 174(T) could be distinguished from its closest relative, P. ingrahamii, by its utilization of D-mannose and D-xylose as sole carbon sources, its ability to ferment myo-inositol and its inability to use fumarate and glycerol as sole carbon sources. In addition, strain 174(T) contained gas vacuoles of two distinct morphologies and grew at temperatures ranging from below 0 to 10 degrees C and its optimal NaCl concentration for growth was 3.5 %. The DNA G+C content was 40 mol%. Whole-cell fatty acid analysis showed that 16 : 1omega7c and 16 : 0 comprised 44.9 and 26.4 % of the total fatty acid content, respectively. The name Psychromonas boydii sp. nov. is proposed for this novel species, with strain 174(T) (=DSM 17665(T) =CCM 7498(T)) as the type strain.

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish.

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from (15)N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are alpha-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. (15)N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.

Psychromonas ingrahamii sp. nov., a novel gas vacuolate, psychrophilic bacterium isolated from Arctic polar sea ice.

A gas vacuolate bacterium, designated strain 37T, was isolated from a sea ice core collected from Point Barrow, Alaska, USA. Cells of strain 37T were large (6-14 microm in length), rod-shaped, contained gas vacuoles of two distinct morphologies, and grew well at NaCl concentrations of 1-10 % and at temperatures of -12 to 10 degrees C. The DNA G+C content was 40 mol%. Whole-cell fatty acid analysis showed that 16 : 1omega7c comprised 67 % of the total fatty acid content. Phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium was closely related to members of the genus Psychromonas, with highest sequence similarity (96.8 %) to Psychromonas antarctica. Phenotypic analysis differentiated strain 37T from P. antarctica on the basis of several characteristics, including cell morphology, growth temperature range and the ability to hydrolyse polymers. DNA-DNA hybridization experiments revealed a level of relatedness of 37 % between strain 37T and P. antarctica, providing further support that it represents a distinct species. The name Psychromonas ingrahamii sp. nov. is proposed for this novel species. The type strain is 37T (=CCUG 51855T=CIP 108865T).

Methylosarcina lacus sp. nov., a methanotroph from Lake Washington, Seattle, USA, and emended description of the genus Methylosarcina.

An obligately methanotrophic bacterial strain, LW14T, isolated from the sediment of Lake Washington, Seattle, USA, is described taxonomically. The isolate is an aerobic, Gram-negative, non-motile bacterium capable of growth on methane, and possesses type I intracytoplasmic membranes (i.e. it is a type I methanotroph). The strain possesses particulate methane monooxygenase (MMO) and has no soluble MMO. Formaldehyde is assimilated via the ribulose monophosphate cycle. The isolate grows within a pH range of 4-8, with the optimum between pH 5.5 and 6.5. The cellular fatty acid profile is dominated by C(16 : )omega18c, C(16 : 1)omega7c and C(16 : 1)omega5t fatty acids. The DNA G+C content is 53.3+/-0.4 mol%. On the basis of sequence analysis of the 16S rRNA gene, isolate LW14T is related most closely to representatives of the genus Methylosarcina. However, DNA-DNA hybridization analysis reveals only a distant relationship between isolate LW14T and the previously described Methylosarcina species. On the basis of its phenotypic and genotypic characteristics, LW14T represents a novel species of the genus Methylosarcina, for which the name Methylosarcina lacus sp. nov. is proposed, with LW14T (=ATCC BAA-1047T=JCM 13284T) as the type strain.

Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate.

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 beta-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both beta-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ beta-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 beta-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.

Analysis of sMMO-containing type I methanotrophs in Lake Washington sediment.

Methane-oxidizing bacteria (methanotrophs) containing soluble methane monooxygenase (sMMO) are of interest in natural environments due to the high co-metabolic activity of this enzyme with contaminants such as trichloroethylene. We have analysed sMMO-containing methanotrophs in sediment from a freshwater lake. Environmental clone banks for a gene encoding a diagnostic sMMO subunit (mmoX) were generated using DNA extracted from Lake Washington sediment and subjected to RFLP analysis. Representatives from the six RFLP groups were cloned and sequenced, and all were found to group with Type I Methylomonas mmoX, although a majority were divergent from known Methylomonas mmoX sequences. Direct hybridization of Lake Washington sediment DNA was carried out using a series of sMMO- and Methylomonas-specific probes to assess the significance of these sMMO-containing Methylomonas-like strains in the sediment. The total sMMO-containing population and the sMMO-containing Methylomonas-like population were estimated to be similar to previous estimates for total methanotrophs and Type I methanotrophs. These results suggest that the major methanotrophic population in Lake Washington sediment consists of sMMO-containing Methylomonas-like (Type I) methanotrophs. The whole-cell TCE degradation kinetics of such a strain, LW15, isolated from this environment, were determined and found to be similar to values reported for other sMMO-containing methanotrophs. The numerical significance of sMMO-containing Methylomonas-like methanotrophs in a mesotrophic lake environment suggests that these methanotrophs may play an important role in methanotroph-mediated transformations, including co-metabolism of halogenated solvents, in natural environments.

Estimation of methanotroph abundance in a freshwater lake sediment.

The numbers of methane-oxidizing bacteria (methanotrophs) in the sediments of Lake Washington were estimated using three culture-independent methods. Quantitative slot-blot hybridizations were performed with type I and type II methanotroph-specific probes. These data were compared to data from quantitative hybridizations using a pmoA-specific probe and a eubacterial probe. From the combined hybridization data, the methanotroph population in Lake Washington was estimated to be 3.6 x 10(8)-7.4 x 10(8) cells/g dry weight. Methanotroph community structure and number were also investigated using polar lipid fatty acid (PLFA) analysis. Analysis of biomarker PLFAs characteristic of both type I (16:1 omega 8) and type II (18:1 omega 8) methanotrophs was used to estimate the abundance of these bacteria in Lake Washington sediments. From the PLFA data, the methanotroph population in Lake Washington was estimated to be 7.1 x 10(8)-9.4 x 10(9) cells/g dry weight. As a third method of quantitation, we calculated the methanotroph population using the total methane oxidation rate for whole cells in Lake Washington sediment to be 1.3 x 10(8)-1.2 x 10(9) cells/g dry weight. The three independent estimates of the number of methanotrophs in Lake Washington sediment agree within a two- to fourfold range. These data suggest that the three techniques used in this study detect the functionally significant population of methanotrophs in Lake Washington. Furthermore, these techniques will be useful for obtaining estimates of methanotroph abundance in additional environments.